1. Field of the Invention
The present invention relates to methods for improving C4 dicarboxylic acid production in filamentous fungi.
2. Description of the Related Art
Organic acids have a long history of commercial use in a variety of industries. For example, organic acids are used in the food and feed industries (citric acid, ascorbic acid, lactic acid, acetic acid, and gluconic acid) as monomers for the production of various polymers (adipic acid, lactic acid, acrylic acid, and itaconic acid), as metal chelators (gluconic acid), and as “green” solvents (acetic acid) (Sauer et al., 2008, Trends in Biotechnology 26: 100-108). Organic acids may themselves be commercial products or they may be chemical building blocks used in the manufacture of other chemicals. In addition to specialty applications, it has long been recognized that C4 dicarboxylic acids can also serve as building block compounds for the production of large volume industrial chemicals, such as 1,4-butanediol, tetrahydrofuran, and gamma-butyrolactone. The cost of producing these large volume industrial chemicals by traditional petrochemical routes has increased significantly due to the high cost of petroleum derived building blocks.
Organic acids are produced commercially either by chemical synthesis from petroleum derived feedstocks (e.g., fumaric acid, malic acid, acrylic acid, and adipic acid) or by microbial fermentation (e.g., citric acid, lactic acid, gluconic acid, and itaconic acid). Some organic acids such as fumaric acid and malic acid can also be produced by microbial fermentation, but are currently produced commercially by chemical synthesis from petrochemical feedstocks due to lower production costs. However, the rising cost of petroleum derived building block chemicals, the geopolitical instability affecting crude oil prices, and the desire to implement manufacturing processes that utilize feedstocks derived from renewable resources have stimulated a renewed interest in producing organic acids and other chemicals by microbial fermentation.
While malic acid is produced commercially today by chemical synthesis from petrochemical feedstocks, it can also be produced by microbial fermentation. Malic acid has been produced at high levels in genetically engineered yeast (Saccharomyces cerevisiae) (Zelle et al., 2008, Appl. Environ. Microbiol. 74: 2766-2777) and naturally occurring filamentous fungi such as Aspergillus spp. (U.S. Pat. No. 3,063,910; Bercovitz et al., 1990, Appl. Environ. Microbiol. 56: 1594-1597). Abe et al. (U.S. Pat. No. 3,063,910) and Bercovitz et al. (1990, Appl. Environ. Microbiol. 56: 1594-1597) reported high levels of malic acid production in several species of Aspergillus. Moreover, Battat et al. (1991, Biotechnol. Bioengineering 37: 1108-1116) reported malic acid production as high as 113 g/L by Aspergillus flavus in a stirred fermentor under optimized conditions. Dicarboxylic acid production by microbial fermentation in yeast is described in WO 2010/003728. Malic acid production by microbial fermentation is also described in WO 2009/011974 and WO 2009/155382. Improvement of malic acid production by genetic engineering of Aspergillus will enable economical commercial malic acid production by fermentation.
Malic acid overproduction in Aspergillus spp. occurs under specific culture conditions (aerobic conditions and high C:N ratio; calcium carbonate is also added as a neutralizing agent and as source of CO2 for malic acid biosynthesis). Under these conditions, overflow metabolism via the cytosolic, reductive tricarboxylic acid (TCA) cycle results in increased malic acid biosynthesis and secretion into the culture medium. Increased malic acid production has been reported in Saccharomyces cerevisiae by increasing the level of pyruvate carboxylase (Bauer et al., 1999, FEMS Microbiol Lett. 179: 107-113) or malate dehydrogenase (Pines et al., 1997, Appl. Microbiol. Biotechnol. 48: 248-255) using genetic engineering and increasing expression of a malic acid transporter (Zelle et al., 2008, supra. It has been suggested, based on biochemical evidence, that malate dehydrogenase activity is limiting malic acid production in Aspergillus flavus strain ATCC 13697 (Peleg et al., 1988, Appl. Microbiol. Biotechnol. 28: 69-75). However, no direct improvement in malic acid production has been shown in Aspergillus as a result of genetic engineering using recombinant DNA techniques.
The present invention relates to methods for improving C4 dicarboxylic acid production, such as malic acid production, in filamentous fungi.